Serosurvey and associated risk factors for Neospora caninum infection in Egyptian water buffaloes (Bubalus bubalis)

Neosporosis is a parasitic disease that causes reproductive disorders in animals, making it a barrier to maximum efficiency. The purpose of this study was to determine the seroprevalence of Neospora caninum (N. caninum) antibodies in water buffaloes from four governorates in northern Egypt. A commercial indirect-ELISA test was used to detect antibodies against N. caninum in the serum of 450 water buffaloes. The total seroprevalence of N. caninum in water buffaloes from Egypt was 31.3%, and the highest prevalence was observed in Gharbia governorate. The identified risk factors for N. caninum infections in water buffaloes were sex (OR = 1.96, 95%CI: 1.22−4.17), buffaloes more than 4 years of age ( OR = 5.80, 95%CI: 2.26−14.86), abortion in second trimester (OR = 16.48, 95%%CI: 2.99−34.03), history of abortion (OR = 3.45, 95%CI: 1.58−7.52) and contact with dogs (OR = 2.55, 95%CI: 1.51−4.32). Thus, more studies are needed to determine the role of buffaloes in the epidemiology of neosporosis in Egypt.


Ethical statement
The Benha University ethics committee for animal experiments approved all methods including the handling and collection of blood samples.In addition, all methods were performed in accordance with the relevant guidelines and regulations.Owners of the buffaloes gave their explicit consent for the collection of the samples.The ARRIVE criteria were adhered to throughout the study process.
Kafr ElSheikh, Qalyubia and Gharbia situated at Nile Delta of Egypt, which characterized by moderate weather at summer and the average annual temperature is 28 °C (with a range of 17 °C to 42 °C), and its average annual precipitation is between 100 to 200 mm.In addition, the prevailing north wind, blowing across the Mediterranean, gives Alexandria a less harsh climate than the surrounding desert.

Study design and sample size
A cross-sectional study was performed during September 2020 to August 2021 to determine the prevalence of antibodies against N. caninum in water buffaloes in the four studied governorates.Using Epi Info™ 7, a representative sample size of 450 buffaloes was determined with a 95% confidence interval, a 5% statistical error, and a 42.8% expected seroprevalence for N. caninum 16 .A total of 450 water buffaloes of various ages and both sexes were chosen at simple random technique from individual animal raised by individual farmers.All of buffaloes included in the study were apparent healthy without in clinical signs and some of them had history of abortion.
Blood samples (5 mL) were obtained from the jugular vein, centrifuged at 3000 rpm for 10 min, serum separated, and kept at −20 °C.An epidemiology questionnaire was used at the time of sampling to collect data from each farmer on the study region, age, sex, breeding system (household or farming system), time of the abortion, previous abortions, and contact with dogs.The information about history and number of abortion or contact with dogs depends on data collected from farmers.

Serological analysis
The antibodies against N. caninum were detected in all sera using commercial indirect ELISA Kit (IDEXX Laboratories Inc., Westbrook, USA).The sensitivity of this kit is 100%, while the specificity 98.9%. the optical density (OD) was measured at 650 nm using microplate ELISA reader (AMR-100, AllSheng, China).Serum samples considered to be positive for N. caninum if their sample to positive (S/P) ratios were more than or equal to 0.50.

Data analysis
Data were collected and analyzed using SPSS ver.24 program (IBM, USA).A univariate analysis of the variables of interest was carried out using the Pearson's chi-squared test to determine factors related to N. caninum infection.The result of P-value < 0.05 was regarded as statistically significant.The results were analysed through the use of univariable logistic regression to evaluate the association between each variable and prevalence of N. caninum in buffaloes.A logistic regression analysis was used to determine the relationship between the existence of anti-N.caninum antibodies and risk variables (sex, age, time and history of abortion, and contact with dogs).The multivariate regression model was used to assess odds ratios (ORs), and confidence intervals (CIs) of each significant variable 20,21 .Odds ratios more than one indicated an increased risk of N. caninum seroprevalence, whereas odds ratios less than one indicated a lower risk of N. caninum seroprevalence.The model's fit was evaluated using the Hosmer and Lemeshow goodness test.

Results
We examined 450 buffalo sera from four Egyptian governorates between September 2020 and August 2021, and the findings revealed that 31.1% (141/450) of examined animals had antibodies against N. caninum, with nonsignificant variation (P = 0.238) between studied governorates, Table 1.www.nature.com/scientificreports/Several factors including sex, age, breeding system, time of abortion, history of abortion and contact with dogs were analyzed to determine the risk factors related with N. caninum infection in water buffalo (Table 2).
There was significant difference (P < 0.05) in seroprevalence in relation to sex and age of examined water buffaloes.The seroprevalence of N. caninum was greater in females (35.9%) than males (11.8%), and in elder buffaloes over 4 years old (53.2%) as compared to young buffaloes under 2 years (17.3%) or buffaloes aged 2-4 years (25.6%).Furthermore, the seroprevalence of N. caninum was significantly (P < 0.05) higher in buffaloes with a history of abortion (59.8%), particularly those aborted in the second trimester (81.5%).In the current investigation, the seroprevalence of N. caninum in buffaloes kept in touch with dogs was substantially greater (38.9%) than in other animals kept away from dogs, Table 1.

Discussion
Infection with N. caninum causes retained fetal membranes and abortion, and subsequent infections raise the risk of reproductive disorders in water buffalo, which causes significant financial losses to the agricultural sector 5,22,23 .However, the available data on the prevalence of associated risk factors for N. caninum infections in Egyptian water buffaloes are scarce.
The seroprevalence rates of N. caninum in buffaloes varied across countries due to a variety of factors such as climatic factors, where the mild temperature and humidity help in growth of oocyst of Neospora 5,37,38 .In addition, the rearing systems, geographic location, and farm management were varied in different countries 15,37,[39][40][41][42][43][44] .Furthermore, comparing studies is challenging since researchers use varied sample criteria, serological procedures, and cut-off values, particularly for the immunofluorescence antibody test 24 .
In the line of previous results of Baltazar-Pérez, et al. 31 , the sex is significant risk factor for prevalence of N. caninum in buffaloes.Contrary to findings of Campero, et al. 35 and Kengradomkij, et al. 27 , the results revealed strong association between N. caninum seroprevalence in water buffaloes and sex.According to our findings, vertical transmission is frequently thought to be the primary mode of infection in bovines 45 .
According to age group, adult animals in our study had a much greater prevalence than young animals.Similarly, several studies have been found significant association between age and the seropositivity for neosporosis in water buffaloes 14,[46][47][48][49] .Indeed, elder age would suggest a possibly longer interaction with polluted water and/ or feed, explaining the increased occurrence.It has been proposed that the age correlation merely reflects the annual increase in the likelihood of being exposed to parasite oocysts.In comparison to younger animals, older animals are predicted to have a larger likelihood of cumulative exposure to infectious agents.In contrast, the seroprevalence of N. caninum in water buffaloes from northern Brazil were not affected by age factor 33,41,[50][51][52] .Interestingly, calves had a significant seroprevalence rate (17.3%), the persistence of maternal antibodies or trans-placental transmission 47 might be to blame for this.According to Cardoso, et al. 53 , maternal antibodies against N. caninum can last up to 21 weeks after birth.
In contrast to the findings of Bărburaș, et al. 46 , we observed no significant relationship between breeding system and N. caninum seroprevalence.The seroprevalence rate was greater in farm animals than in house-hold www.nature.com/scientificreports/animals, which was due to the chance of farm animals being exposed to sources of parasite oocyst contamination being higher.
There is no information in Egypt about buffalo reproductive losses caused by N. caninum infection.The findings of this study revealed strong association between N. caninum infection and abortion especially in second trimester stage.These findings were consistent with what have been found in previous studies of Romero-Salas, et al. 30,54 , Anderson, et al. 55 and Wouda, et al. 56 .This provides circumstantial evidence that N. caninum may have a role in water buffaloes abortions in the research region.
Similarly, to the findings of Oliveira, et al. 24 , N. caninum prevalence rate was greater among water buffaloes kept with close contact with pet dogs.This could be attributed to dogs consuming aborted materials and consequently shedding infective oocyst which contaminate food and play a significant role in horizontal spreading of N. caninum infection to vulnerable animals 57 .
The limitation of this study is random sampling because it does truly represent about the prevalence of the disease in the studied areas.

Conclusion
The present findings confirmed that presence of antibodies against N. caninum in water buffaloes in studied governorates in Northern Egypt.In addition, some factors such as sex, age, time or history of abortion and contact with dogs considered as potential risk factors for N. caninum infection.These findings highlight the relevance of N. caninum control and prevention in Egypt, where water buffaloes are major domestic animals, as well as the need of risk factor analysis for efficient neosporosis control in various locations.The effects of Neospora infections in water buffalo should be further studied, but it's also important to look at the epidemiological significance of sick buffaloes and their connection to bovine neosporosis.

Figure 1 .
Figure 1.Map showed the governorates under the study (map generated by QGIS program).

Table 1 .
Seroprevalence of N. caninum in water buffaloes in relation to different studied factors.*The results considered significant if P-value less than 0.05.

Table 2 .
Multivariate logistic regression analysis for variables associated with seroprevalence of N. caninum in water buffaloes.B logistic regression coefficient, SE standard error, OR odds ratio, CI confidence interval.